Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The specificity determinants for insulin-stimulated protein kinase-I (ISPK-1) have been investigated with synthetic peptides based on naturally-occurring protein phosphoacceptor sequences. Peptides (Arg-Arg-Xaa-Ser-Xaa) that fulfill the consensus sequence for cyclic-AMP-dependent protein kinase (PK-A) are also phosphorylated readily by ISPK-1. The phosphorylation efficiency is improved by increasing the number of N-terminal arginine residues and by moving the arginyl cluster one residue further away from the serine, the nonapeptide (Arg)4-Ala-Ala-Ser-Val-Ala being the best substrate among all the short peptides tested (Km = 15 microM). Conversely, the substitution of either Thr for Ser or Lys for Arg is detrimental. Likewise, two flanking Pro residues and an Arg immediately N-terminal to the Ser act as negative specificity determinants. While the specificity of ISPK-1 shows several similarities to that of PK-A, including an absolute requirement for basic residues on the N-terminal side of the target Ser, it differs in several other respects including (1), the detrimental effect of a Lys for Arg substitution which is still compatible with some phosphorylation by ISPK-1, but not PK-A; (2), the presence of C-terminal acidic residues which are tolerated very well by ISPK-1, but are detrimental to PK-A; (3), the effect of substituting Phe for Val in the peptide Arg-Arg-Ala-Ser-Val-Ala, which improves the efficiency of phosphorylation by PK-A (lowering the Km 4-fold), but has no effect on phosphorylation by ISPK-1. These differences in peptide substrate specificity may account in part for the different rates of phosphorylation of physiological substrates for ISPK-1 and PK-A, such as the G subunit of protein phosphatase-1.
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PMID:An analysis of the substrate specificity of insulin-stimulated protein kinase-1, a mammalian homologue of S6 kinase-II. 834 77

In this report, we show that the p14 subunit of calcium-binding myeloid protein complex (p8,14) is phosphorylated in human neutrophils stimulated with either fMet-Leu-Phe, phorbol myristate acetate, or a calcium ionophore. Trifluoperazine, a calmodulin antagonist, caused hyperphosphorylation of p14 in intact resting neutrophils. Preincubation of resting cells with 10-20 nM calyculin A, a potent protein phosphatase inhibitor, also caused enhanced labeling of p14, which was further progressively increased on stimulation with fMLP. Thus, the phosphorylation level of p14 in resting as well as in stimulated neutrophils appears to be controlled by an active protein phosphatase. The phosphorylation of p14 by a chemoattractant and by a phorbol ester is a novel finding supporting the current belief that p8,14 myeloid protein may play an important role in the metabolism of myeloid cells.
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PMID:Calcium-binding myeloid protein (P8,14) is phosphorylated in fMet-Leu-Phe-stimulated neutrophils. 836 May 91

Previously employed non-selective protein kinase inhibitors yielded inconclusive results regarding involvement of protein kinase C (PKC) in phosphorylation of 47 kDa protein (p47 phox) in intact neutrophils stimulated with physiologic agonists of superoxide generation. In the present study, phosphorylation of p47 phox in formylMet-Leu-Phe (fMLP) stimulated neutrophils was potently inhibited in the presence of 0.3 microM RO 31-8220, a selective inhibitor of PKC. These results provide experimental evidence in support of the currently considered essential involvement of PKC in p47 phox phosphorylation in response to physiologic stimulation of neutrophil surface receptors. The fMLP-induced phosphorylation of p47 phox was enhanced and prolonged by calyculin A, a specific inhibitor of protein phosphatases of types 1 and 2A, and such enhanced phosphorylation was also effectively inhibited by RO 31-8220. Our results suggest that the extent and duration of p47 phox phosphorylation in intact fMLP-stimulated neutrophils is probably controlled by a balance between the activities of PKC, on the one hand, and of protein phosphatase(s) of type(s) 1 and/or 2A, on the other. Effects of RO 31-8220 and of calyculin A on the fMLP-induced p47 phox phosphorylation were paralleled by similar effects on superoxide release. Calyculin A and RO 31-8220 were also used to study signal transduction by a post-receptor agonist of superoxide generation, a calcium ionophore A23187. The results of the latter study indicated that PKC was activated in A23187-stimulated neutrophils and was essentially involved in superoxide generation and p47 phox phosphorylation. Further, these results suggested that protein phosphatase(s) of type(s) 1 and/or 2A were also activated in A23187-signalling pathway, and limited the extent of superoxide release and p47 phox phosphorylation.
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PMID:Involvement of protein kinase C and of protein phosphatases 1 and/or 2A in p47 phox phosphorylation in formylmet-Leu-Phe stimulated neutrophils: studies with selective inhibitors RO 31-8220 and calyculin A. 851 1

Recent studies have shown that substitution of Ala for one or more Phe residues in calmodulin (CaM) imparts a temperature-sensitive phenotype to yeast (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). The Phe residue immediately preceding the first Ca(2+) ligand in site III of CaM (Phe-92) was found to be of particular importance because the mutation at this position alone was sufficient to induce this phenotype. In the present work we have studied the functional and structural consequences of the Phe-92 --> Ala mutation in human liver calmodulin. We found that the mutant (CaMF92A) is incapable of activating phosphodiesterase, and the maximal activation of calcineurin is reduced by 40% as compared with the wild type CaM. Impaired regulatory properties of CaMF92A are accompanied by an increase in affinity for Ca(2+) at the C-terminal domain. To investigate the structural consequences of the F92A mutation, we constructed four recombinant C-terminal domain fragments (C-CaM) of calmodulin (residues 78-148): 1) wild type (C-CaMW); 2) Ala substituted for Phe-92 (C-CaMF92A); 3) cysteine residues introduced at position 85 and 112 to lock the domain with a disulfide bond in the Ca(2+)-free (closed) conformation (C-CaM85/112); and 4) mutations 2 and 3 combined (C-CaM85/112F92A). The Cys-containing mutants readily form intramolecular disulfide bonds regardless whether Phe or Ala is present at position 92. The F92A mutation causes a decrease in stability of the domain in the absence of Ca(2+) as indicated by an 11.8 degree C shift in the far UV circular dichroism thermal unfolding curve. This effect is reversed by the disulfide bond in the C-CaM85/112F92A mutant. The C-CaMW peptide shows a characteristic Ca(2+)-dependent increase in solvent-exposed hydrophobic surface which was monitored by an increase in the fluorescence of the hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid. The fluorescence increase induced by C-CaMF92A is approximately 45% lower than that induced by C-CaMW suggesting that the F92A mutation causes a decrease in the accessibility of several hydrophobic side chains in the C-terminal domain of CaM in the presence of Ca(2+). The Cys-85-Cys-112 disulfide bond causes a 10- or 5.9-fold decrease in Ca(2+) affinity depending on whether Phe or Ala is present at position 92, respectively, suggesting that coupling between Ca(2+) binding and the conformational transition is weaker in the absence of the phenyl ring at position 92. Our results indicate that Phe-92 makes an important contribution to the Ca(2+)-induced transition in the C-terminal domain of CaM. This is most likely the reason for the severely impaired regulatory properties of the CaM mutants having Ala substituted for Phe-92.
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PMID:The role of Phe-92 in the Ca(2+)-induced conformational transition in the C-terminal domain of calmodulin. 862 80

P1C3 is a monoclonal antibody that binds p19, a novel neutrophil activation antigen that translocates to the cell surface upon neutrophil activation. We find that P1C3 inhibits capacitative Ca2+ entry, induced by emptying the intracellular Ca2+ stores with thapsigargin. The effect is transient, reaching its maximum at 30-60 s, but becomes permanent upon pretreatment of the cells with the protein phosphatase inhibitor calyculin A, suggesting the involvement of protein phosphorylation. The inhibitory action is similar to the one reported previously for the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), although the transduction mechanism may be different. Inhibition of Ca2+ entry by fMLP was prevented by pretreatment with pertussis toxin, whereas inhibition by P1C3 was not. Pretreatment with cholera toxin had no effect. This suggests that the effect of P1C3 may not be mediated by a heterotrimeric G protein. Tyrosine kinase inhibitors did not prevent inhibition by either fMLP or P1C3. Phospholipase C activation seems not to be involved as P1C3, contrarily to fMLP, was unable to induce Ca2+ release from the intracellular Ca2+ stores.
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PMID:Transient inhibition of capacitative calcium entry in human neutrophils by a monoclonal antibody directed against a 19-kDa antigen. 883 Jul 88

We studied the effect of hyperphenylalaninemia on in vitro incorporation of 32P into cytoskeletal proteins from cerebral cortex of rats by injecting l-phenylalanine plus alpha-methylphenylalanine subcutaneously from the 6th to the 14th day postpartum. Chronic hyperphenylalaninemia induced an increased in vitro phosphorylation of the 150-kDa neurofilament subunit and tubulins present in the cytoskeletal fraction at the end of the treatment and 3 days after treatment discontinuation. In addition, when in vitro phosphorylation of the cytoskeletal proteins from treated animals was performed in the presence of the drugs we observed a decreased in vitro incorporation of 32P into these proteins. Thus, the effect of l-phenylalanine plus alpha-methylphenylalanine on the endogenous protein kinase and phosphatase activities was examined and the results demonstrated that these drugs have an inhibitory effect on calcium/calmodulin-dependent protein kinase II and protein phosphatase type 1.
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PMID:Effect of hyperphenylalaninemia chemically induced on in vitro incorporation of 32P into cytoskeletal proteins from cerebral cortex of developing rats. 905 82

The diverse forms of protein phosphatase 1 in vivo result from the association of its catalytic subunit (PP1c) with different regulatory subunits, one of which is the G-subunit (G(M)) that targets PP1c to glycogen particles in muscle. Here we report the structure, at 3.0 A resolution, of PP1c in complex with a 13 residue peptide (G(M[63-75])) of G(M). The residues in G(M[63-75]) that interact with PP1c are those in the Arg/Lys-Val/Ile-Xaa-Phe motif that is present in almost every other identified mammalian PP1-binding subunit. Disrupting this motif in the G(M[63-75]) peptide and the M(110[1-38]) peptide (which mimics the myofibrillar targeting M110 subunit in stimulating the dephosphorylation of myosin) prevents these peptides from interacting with PP1. A short peptide from the PP1-binding protein p53BP2 that contains the RVXF motif also interacts with PP1c. These findings identify a recognition site on PP1c, invariant from yeast to humans, for a critical structural motif on regulatory subunits. This explains why the binding of PP1 to its regulatory subunits is mutually exclusive, and suggests a novel approach for identifying the functions of PP1-binding proteins whose roles are unknown.
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PMID:Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1. 915 14

Application of either acetylcholine (ACh), dopamine (DA), histamine (HA), or Phe-Met-Arg-Phe-NH2 (FMRFamide) induces a K+-current response in the identified neurons of Aplysia under voltage clamp. This type of response is mediated by a pertussis toxin (PTX)-sensitive G-protein, Gi or Go. Extracellular application of 60 microM phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), to these cells markedly depressed all the K+-current responses to ACh, DA, HA, and FMRFamide. The depressing effect of PDBu lasted for at least 60 min despite continuous washing with the normal perfusing medium. Application of PKC inhibitors such as 100 microM H-7 or 10 microM staurosporine and PKCI(19-31) prior to the application of PDBu significantly decreased the depressing effects of PDBu. In contrast, an intracellular injection of okadaic acid (OA), an inhibitor of protein phosphatase 1 and 2A, significantly augmented the blocking effect of PDBu. Intracellular injection of the PKC catalytic subunit induced a similar depressing effect as observed with PDBu. The dose-response curves obtained with different transmitters all shifted downward after the activation of PKC, but the ED50 of each transmitter remained unchanged. Furthermore, the K+-current responses induced by the intracellular application of GTPgammaS were not depressed at all, even after the receptor-induced K+-current responses of the same cell were markedly depressed. These results strongly suggest that PKC phosphorylated a certain coupling site between the receptor and G-protein, and impaired the signal transduction necessary for triggering the K+-channel opening.
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PMID:Functional uncoupling between the receptor and G-protein as the result of PKC activation, observed in Aplysia neurons. 927 Nov 55

The carboxy terminus of protein phosphatase 2A (PP2A) catalytic subunit is highly conserved. Seven out of the last nine residues, including two potential in vivo phosphorylation sites, threonine 304 and tyrosine 307, are completely invariant in all known PP2As. Mutational analysis of the carboxy terminus in vivo was facilitated by efficient immunoprecipitation of trimeric PP2A holoenzyme via an epitope-tagged catalytic subunit. The results indicate that the catalytic subunit carboxy terminus is important for complex formation with the PP2A 55 kDa regulatory B subunit, but not with polyomavirus oncogene, middle tumor antigen (MT), a viral B-type regulatory subunit. Replacing catalytic subunit threonine 304 or tyrosine 307 with a negatively charged amino acid abolished binding of the B subunit to the dimeric enzyme core and altered substrate specificity. Certain other amino acid substitutions of different size and/or charge also abolished or greatly reduced B subunit binding. Substitution of alanine at position 304 or phenylalanine at position 307 did not dramatically reduce B subunit binding or phosphatase activity in vitro, yet the latter substitutions are not found in naturally occurring PP2As. Thus, the wild-type residues are important for a yet unknown function in vivo. Additionally, deleting the carboxy terminal nine amino acids inhibited binding of the B subunit to the dimeric enzyme core, indicating a requirement for one or more of these amino acids for complex formation. MT interaction with the dimeric PP2A enzyme core was not inhibited by any of these mutations. Finally, unlike B subunit, MT does not activate the phosphatase activity of the PP2A heterodimer towards cdc2-phosphorylated histone H1.
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PMID:Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen. 928 86

We have purified a form of protein phosphatase 1 (PP1) from HeLa cell nuclei, in which the phosphatase is complexed to a regulatory subunit termed p99. We report here the cloning and characterisation of the p99 component. p99 mRNA is widely expressed in human tissues and immunofluorescence analysis with anti-p99 antibodies showed a punctate nucleoplasmic staining with additional accumulations within the nucleolus. The C-terminus of p99 contains seven RGG RNA-binding motifs, followed by eleven decapeptide repeats containing six or more of the following conserved residues (GHRPHEGPGG), and finally a putative zinc finger domain. Recombinant p99 suppresses the phosphorylase phosphatase activity of PP1 by > 90% and the canonical PP1-binding motif on p99 (residues 396-401) is unusual in that the phenylalanine residue is replaced by tryptophan.
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PMID:Purification and characterisation of p99, a nuclear modulator of protein phosphatase 1 activity. 945 May 50


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